Real-time polymerase chain reaction was used to develop a one-stage procedure for molecular genetic analysis of Mycobacterium tuberculosis (MBT) DNA in order to determine mutations associated with drug resistance to the antituberculous agents: isoniazid and rifampicin. To analyze the spread of drug-resistance of the causative agent of tuberculosis in Russia, two thousand MBT strains were studied in 24 regions of all the federal districts. Testing 1406 MBT strains isolated by first detected and untreated patients revealed multidrug resistance (MDR) in 21.9% of cases. MRD was detected in 58.5% of the previously treated patients with MDR. The agreement of molecular genetic analysis of drug resistance with the results of cultural tests of 1096 strains was 94%.